Several novel proteins now have been identified which appear to act as growth or differentiation factors, regulating cell proliferation or differentiation. One group of proteins now has been demonstrated to act as true tissue morphogens, competent to induce the proliferation and differentiation of progenitor cells into functional tissue that is properly vascularized and ennervated. These proteins, referred to herein as "morphogens," includes members of the family of bone morphogenetic proteins (BMPs) which were initially identified by their ability to induce ectopic, endochondral bone morphogenesis. The morphogens generally are classified in the art as a subgroup of the TGF-.beta. superfamily of growth factors (Hogan (1996) Genes & Development 10:1580-1594). Members of the morphogen family of proteins include the mammalian osteogenic protein-1 (OP-1, also known as BMP-7, and the Drosophila homolog 60A), osteogenic protein-2 (OP-2, also known as BMP-8), osteogenic protein-3 (OP-3), BMP-2 (also known as BMP-2A or CBMP-2A, and the Drosophila homolog DPP), BMP-3, BMP-4 (also known as BMP-2B or CBMP-2B), BMP-5, BMP-6 and its murine homolog Vgr-1, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, GDF-5, GDF-6, and GDF-7 (also known respectively as CDMP-1, CDMP-2, and CDMP-3), and the Xenopus homolog Vgl. Members of this family encode secreted polypeptide chains sharing common structural features, including processing from a precursor "pro-form" to yield a mature polypeptide chain competent to dimerize, and containing a carboxy terminal active domain of approximately 97-106 amino acids. All members share a conserved pattern of cysteines in this domain and the active form of these proteins can be either a disulfide-bonded homodimer of a single family member, or a heterodimer of two different members (see, e.g., Massague (1990) Annu. Rev. Cell Biol. 6:597; Sampath, et al. (1990) J. Biol. Chem. 265:13198).
The members of the morphogen family of proteins are expressed in a variety of tissues during development, as well as in adult tissues. BMP-3 for, example, is expressed in developing human lung and kidney (Vukicevic et al. (1994) J. Histochem. Cytochem, 42:869-875), and BMP-4 is expressed in the developing limbs, heart, facial processes and condensed mesenchyme associated with early whisker follicles in embryonic mice (Jones, et al. (1991) Development 111:531-542). OP-1 (BMP-7) expression is identified in basement membranes in human embryos, including those of the developing lungs, pancreas, skin, and convoluted tubules of kidneys, ocular tissues, developing brain, and in a variety of developing neural origin tissues (Vukicevic, et al. (1994) Biochem. Biophys. Res. Commun, 198:693-700) and WO 92/15323 (PCT/US92/01968). Similarly, Vgr-1, the murine BMP-6 homolog, is expressed most abundantly in adult lung and to a lesser extent, in adult kidney, heart and brain. Some of the morphogens (e.g., OP-2) have not yet been detected in analyses of adult tissues, suggesting only an early developmental role for these morphogens (Ozkaynak, et al. (1992) J. Biol. Chem. 267:25220-25227). In contrast, high levels of murine OP-1 expression have been observed in adult mouse kidneys (Ozkaynak, et al. (1991) Biochem. Biophys. Res. Commun. 179:116-123). Thus, OP-1 synthesized in the kidney likely can act as a paracrine regulator of bone growth, which would be consistent with the role of the kidneys in both calcium regulation and bone homeostasis. See, for example, WO 92/15323 (PCT/US92/01968, published Mar. 11, 1992) and WO 93/05172 (PCTIUS92/07359, published Mar. 18, 1993).
Two morphogens of particular interest are OP-1 and BMP-4. These proteins, along with their Drosophila homologs, are competent to induce the chondroblastic or osteoblastic phenotype in vitro and to induce endochondral bone formation in vivo, including formation of cartilage, bone and bone marrow, including articular cartilage. Recent studies have revealed that, in general, members of this BMP family have a wide variety of effects not only on bone but on many other cell types, e.g., induction of ventral mesoderm tissues in embryo including renal and adrenal tissues, liver, spleen, pancreas, and skin; induction of neural cells, including retinal cells, cells of the substantial nigra and cerebral cortex, neural cells of central and peripheral nervous system; and initiation of epithelial mesenchymal interactions in tooth development including dentin, cementum and periodontal ligament tissues See, for example, WO 92/15323 (PCT/US92/01968 published Mar. 11, 1992), WO 93/04692 (PCT/US92/07358 published Mar. 18, 1993), WO 94/03200 (PCT/US93/07231 published Feb. 17, 1994), WO 94/06449 (PCT/US93/08808 published Mar. 31, 1993), WO 94/06399 (PCT/US93/08742 published Mar. 31, 1994), and WO 94/06420 (PCT/US93/08885 published Mar. 31, 1994).
The BMP family of proteins are believed to be competent to induce true morphogenesis of mammalian tissue. As a result, significant effort has been devoted to characterizing and developing these and other functionally and structurally related proteins for use in the regenerative healing of injured or diseased mammalian tissues or organs. Particular effort has been devoted to developing morphogen-based therapeutics for the treatment of injured or diseased mammalian bone, dental, periodontal, renal, hepatic and neural tissues, including for example, therapeutic compositions for inducing regenerative healing of bone defects such as fractures, as well as for preserving or restoring healthy metabolic properties in diseased bone tissue, e.g., osteopenic bone tissue. Complete descriptions of efforts to develop and characterize morphogen-based therapeutics for use in mammals, including humans, are set forth in pending U.S. patent application Ser. Nos. 08/404,113, 08/396,930, 08/445,467, 08/152,901, abandoned, Ser. Nos. 08/432,883, 08/155,343, now U.S. Pat. No. 5,656,593, Ser. Nos. 08/260,675, 08/165,541, abandoned, Ser. No. 08/174,605, abandoned, and Ser. No. 07/971,091, abandoned, the teachings of each of which are incorporated herein by reference.
Presently certain complications, however, are encountered during the production, formulation and use in vivo of therapeutic macromolecules, such as morphogen proteins. For example, such proteins are typically produced by fermentation or culture of suitable host cells. Any biological product produced from such host cells for use in humans presently must be shown to be essentially free of host cell contaminants, such as secreted or shed proteins, viral particles or degradation products thereof. Providing such assurance can add significantly to the cost and technical difficulty of commercial production of biological macromolecules. Furthermore, appropriate formulations must be developed for conferring commercially reasonable shelf life on the produced macromolecule, without significant loss of biological efficacy. In addition, useful dosages for administration to an individual need to be determined for each tissue application. For example, in periodontal restoration administering excess BMP2 resulted in bone formation in addition to partial regeneration of periodontium in at least one instance. (Sigurdsson et al. (1995) J. Periodontal 66:131-138). An additional complicating factor arises when circumstances warrant an extended course of therapeutic treatment: the treated mammal can develop an immunological response to the macromolecule, and any such response can interfere with effectiveness thereof. In extreme circumstances, treatment must be discontinued.
Accordingly, needs remain for the identification of therapeutically effective analogs of the aforesaid morphogens, particularly for analogs that are inexpensive to produce, are robust upon storage, and have a reduced propensity for eliciting undesirable side effects upon chronic or repeated administration to a mammal.
Objects of the present invention are to (1) provide novel biological markers of tissue morphogenesis including chondrocyte and osteoblast differentiation and bone tissue morphogenesis; (2) provide novel biological markers which appear early in the cascade of events defining morphogen induced morphogenesis; (3) identify novel inducers of morphogenesis that appear downstream of the BMP catalyzing effect; and (4) to provide novel therapeutics derived from these downstream inducers. Still another object of the invention described herein is to provide methods and compositions for identifying a morphogen analog, that is, for identifying a substance that mimics in part or whole a morphogen-induced biological effect in living cells or tissue. Another object of the present invention is to provide an analog identified according to the present identification method. Yet another object is to provide a therapeutic composition comprising an identified analog suitable for administration to a mammal in need thereof, such as a mammal afflicted with a metabolic bone disease, e.g., a disease characterized by osteopenia. Still another object is to provide a bench-mark useful in assessing proper doses of morphogens or analogs thereof.
The foregoing and other objects and features of the invention will be apparent from the description, drawings and claims which follow.